California Department of Food and Agriculture PD/GWSS
Final Report
July 2012
Report title: Final Report for CDFA Agreement Number 03-0293 Amend 4
Project Title: Breeding Pierce’s disease resistant winegrapes.
Principal Investigator and Cooperating Staff: M. Andrew Walker and Alan Tenscher, Dept. of Viticulture & Enology, University of California, One Shields Ave., Davis, CA 95616-8749, , 530-752-0902
Reporting period: July 2003 to July 2012
Introduction
The Walker lab has a long history of breeding for PD resistance having developed rapid screening techniques for Xylella fastidiosa (Xf) resistance (Buzkan et al. 2003, Buzkan et al. 2005, Krivanek et al. 2005a 2005b, Krivanek and Walker 2005), and possessing unique and highly resistant V. rupestris x V. arizonica selections, as well as an extensive collection of southern US grape hybrids, to allow the introduction of extremely high levels of Xf resistance into commercial grapes. We have genetically mapped PD resistance from V. arizonica/candicans b43-17 and have identified very tightly linked markers for use in marker-assisted selection (MAS) (Krivanek et al. 2006, Riaz et al. 2006, Riaz et al. 2007, Riaz et al. 2008, Riaz et al. 2009). We have used these markers to rapidly select at each generation of the backcross breeding program as we increase the percentage of vinifera in the hybrid progeny (F1 = 50% vinifera; backcross (BC) 1 = 75%; BC2 = 87%; BC3 = 94%; BC4 = 97% vinifera). Using aggressive training and growing practices we have been able to force a large portion of each generation to flower in the second year, thus reducing the seed-to-seed generation time to two years. In 2009 we produced about 12,000 seeds at the BC4 97% vinifera generation; about half of these germinated and about half of the resulting progeny had markers for PdR1. About 2,000 plants were planted from this generation and we began selecting them for fruit quality in the Fall of 2011. The goal has been to introgress PdR1 into the major winegrape variety types to produce new resistant winegrapes that are similar to internationally recognized, high quality winegrapes. We made crosses to produce about the same number of PdR1 97% vinifera in 2010 and 2011 and these will fruit in 2012 and 2013, respectively.
The best selections are greenhouse screened so that only the selections with the strongest PD resistance are advanced to wine quality testing. We then propagate the best selections to produce 6 to 8 vine replicates that can be used for micro-vinification. This process has been used to produce PD resistant wines at the 94% (BC3) vinifera level and these micro-scale wines have scored well in tasting panels; significantly better than those at the 88% vinifera level. The best of the 97% vinifera selections will then be multiplied for 100 vine commercial scale testing prior to release.
The next phase of the breeding program will be to incorporate resistance from other sources. PdR1 is a single gene resistance, which increases its chances of breaking down. We are studying other sources of resistance, both single and multiple gene, and have begun the process of crossing these resistance sources into the PdR1 lines. The breeding program also screens potential parents and genetic mapping populations for a companion project – “Genetic mapping of Xylella fastidiosa resistance gene(s) in grape germplasm form the southern United States”. This project is critical to the success of the next phase of the PD breeding program as it will produce genetic markers to these new forms of resistance, which will accelerate breeding progress and to allow the various sources of resistance to be combinedintomulit. Without the markers it would be very difficult to determine whether multiple resistances were combined in one background, since the expression of either gene will be very similar – PD resistant plants. In addition to multiple forms of PD resistance we are also making crosses to introduce powdery mildew resistance into our advanced PD resistant lines. Finally, some of the resistant selections host very low levels of X. fastidiosa in their xylem. These plants do not express disease, but if the bacteria moved down into the rootstock it might kill the vine. This possibility stimulated the production of rootstocks with PdR1 and good resistance to a wide range of nematodes. If the PD resistant selections are grafted on these rootstocks they will be resistant from the roots to the shoot tips.
Over the past 10 years, this breeding program has made hundreds of crosses, produced tens of thousands of seeds, planted and trained thousands of seedlings, and greenhouse tested thousands of plants. It is now ready to begin field and wine testing leading to release of new varieties and to strengthen resistance by complexing multiple forms into one line.
Objectives
1. Breed PD resistant winegrapes through backcross techniques using high quality V. vinifera winegrape cultivars and Xf resistant selections and sources characterized from our previous efforts.
2. Continue the characterization of Xf resistance and winegrape quality traits (color, tannin, ripening dates, flavor, productivity, etc.) in novel germplasm sources, in our breeding populations, and in our genetic mapping populations.
Results – Research highlights
2003 - 2004 – Many crosses were made and progeny tested to examine resistance in a range of southern Vitis species and southeastern US breeder selections. Table 1 displays the range of crosses made. In this phase of the project we were shifting focus from table grapes to wine grapes. We also made the first crosses to produce PD resistant rootstocks.
2004 - 2005 –We increased the number of seedlings and high fruit quality selections we test under our greenhouse screen. This screening is very severe, but material that passes the screen is reliably resistant and dramatically restricts Xylella fastidiosa (Xf)movement. We are also co-screening for powdery mildew resistance. The heritability of Xf resistance from a range of resistant southeast US (SEUS) cultivar and species parents is not consistent – some parents produce few resistant offspring, while others produce a large percentage – making careful parental screening very important. MAS for PdR1 is now being applied to seedling populations from PdR1resistant parents (Table 2).
2005 - 2006 – Marker-assisted selection is reliably being used for PdR1 resistance. BC2 (88% vinifera)have been produced. In this year we attempted to streamline the greenhouse resistance screen so that less space and time would be required to test plants. However, smaller pots and closer spacing did not produce reliable results compared to our wider spacing, 10 cm square pot, 12 to 16 week assay. This year’s crosses produced about 8,500 seeds – many with >75% V. vinifera parentage (Table 3). Evaluation of the 75% vinifera progeny found that the plants were much more vinifera-like in appearance and that they were losing their wild species characteristics (Table 4).
2006 - 2007 –This year’s crosses were focused on broadening the V. vinifera winegrape base in our breeding lines. We produced thousands of seed with 87.5% and 75% vinifera progeny (Table 5). Many of our current populations have the PdR1 allele from F8909-08; we made many crosses this year to include the alternate PdR1 allele from F8909-17. Crosses were also made to produce a new mapping population for a collaborative project with the USDA-Parlier to allow mapping of PD resistance from the resistant Florida selection BD5-117, which will help with comparative evaluations of PD resistance genes. The best sources of PD resistance allow very low levels of Xf to develop in xylem vessels. If these were grafted onto phylloxera resistant rootstocks, the Xf in them might kill the rootstocks. Thus, we made additional crosses to produce a PD and nematode resistant rootstock, and can use MAS for both PdR1 and the Xiphinema index resistance gene, XiR1. A number of 88% vinifera were evaluated for growth habit, foliar appearance, fruit characters and the first wines were also made (Table 6a through 6d). These had Syrah and Chardonnay as their last backcross parent.
2007 - 2008–The 2008 crosses (Table 7) were made to: 1) Use the PdR1 allele from 8909-08 to broaden the vinifera winegrape lines at the 94% vinifera level; 2) Combine PdR1 with the powdery mildew resistance gene Run1 at the 90.6% vinifera level; 3) Combine PdR1 with the LG13 powdery mildew resistance gene REN1 at the 88% vinifera level; 4) Use 8909-17 based resistance with diverse vinifera winegrapes to produce resistant progeny at the 88% vinifera level; 5) Use the F1 progeny of the homozygous PD resistant b40-14 V. arizonica to produce a breeding and mapping population that is 75% vinifera; 6) use elite winegrapes to broaden and expand the V. shuttleworthii breeding lines producing progeny that are 75% and 88% vinifera and 7) Produce rootstocks with PdR1 and broad-based nematode resistance. Inoculations were made to selections with PdR1 at the 88% and 75% vinifera at our Beringer, Napa County trial. Finally, small-scale wine lots were made from five 88% viniferaPdR1 selections from wine grape backgrounds. Fruit evaluation and must analysis were performed on numerous other promising progeny at this level.
2008 - 2009 –The 2009 breeding goals were: 1) create 97% V. vinifera seedlings with PD resistance using PdR1 from V. arizonica hybrid– F8909-08; 2) create 75% V. vinifera seedlings with PD resistance from V. arizonica b40-14 (alternate single gene source of PD resistance; and 3) enlarge the V. arizonica/girdiana b42-26 (multigenic resistance to PD) mapping population by remaking the V. vinifera F2-35 x b42-26 cross. Five hundred and thirty-three seedlings from breeding lines, mapping populations and PD resistant rootstocks were greenhouse screened for Xf resistance. One hundred and seventy-two table and raisin grape selections were also greenhouse screened, with another 175 under testing. Winegrape selections with PdR1 at the 87.5% and 75% vinifera level in our Beringer, Napa County trial were inoculated and a similar trail using selections at the 94% vinifera level was planted. Small-scale wine lots were made from three 94% vinifera and five 87.5% viniferaPdR1 selections. Fruit evaluation and juice analysis were performed on numerous other promising progeny at the 94% vinifera PdR1 level. Finally, 32 crosses with 94% viniferaPdR1 selections yielded about 11,500 seeds, which will sprout in 2010 to produce the 97% vinifera PdR1 generation (Table 8).
2009 - 2010 – The 2010 crosses (Table 9) were made to increase the number of 97% vinifera progeny with PdR1. Seedlings from these crosses will begin fruiting in 2012. We also made crosses to bring PdR1 from b43-17 without the V. rupestris parentage from F8909-08. This effort is now in the BC3 generation and we will make wines from select progeny to determine whether the V. rupestris parentage (associated with very dark juice and strong peppery flavors) has long term detrimental effects to wine quality. We also want to test whether the V. rupestris parentage influences the level of PD resistance. Many selections were greenhouse screened for resistance to PD (Table 10) and wines were again from PdR1 selections at the 88 and 94% vinifera level (Table 11).
2010 - 2011 –The first of the 97% viniferaPdR1 seedlings began fruiting in 2011 (Table 12). This is the generation we plan to select from for release. We selected 77 as worthy of wine testing on a 6 to 8 vine scale and from these we will select for 100 vine commercial scale testing. Crosses were made to BC5 generation PdR1 progeny, to further backcrossing with non-V. rupestris progeny, and to advance resistance from b42-26 an excellent but multi-genic form of PD resistance (Table 13). We planted a new plot in Healdsburg with 94% viniferaPdR1 selections and added new materials to our plot in Yountville, and have distributed cuttings of 87% viniferaPdR1 resistant selections to Texas for a field plot near Fredericksburg. This plot augments trials with the same selections in Auburn, AL, and Galveston, TX. Small-scale wines were made from 94% vinifera PdR1 selections grown in the UCD vineyards and they were better than Chardonnay and Cabernet Sauvignon, and the PD resistant cultivars Lenoir and Blanc du Bois made with the same quantities of fruit.
2011 - 2012 – Table 13 summarizes our PD resistant wine type seedling production, MAS testing and planting from crosses made in 2011. Table 13a details the addition of two new elite vinifera backgrounds into our existing 97-98% vinifera PD resistant groups in the PdR1b line. Since the beginning of our GH screening, we have observed that after greenhouse inoculation V. arizonica/candicans b43-17 always has lower Xf titer than any of its descendants. Table 13b shows two small populations developed to evaluate the b43-17 resistance line in the absence of the major PdR1 resistance locus. In parallel with our continued mapping efforts to identify resistance loci in the b42-26 line, we have been using our greenhouse screen to identify particularly resistant backcross individuals. 07344A-35 is one such genotype having tested highly resistant on three separate occasions; it’s now at the 88% vinifera level (Table 13c). Similarly we took the opportunity to cross this same b42-26 resistant individual with one of our most resistant 97% PdR1b resistant genotypes to create a small test population to evaluate the efficacy of combining these two seemingly different resistant lines (Table 13d).
Table 14a summarizes the estimated number of seeds produced from 10 different crosses that advance the PdR1a allele from b43-17 to the 97% vinifera level. Previously we identified a PD resistance locus PdR1c from V. arizonica b40-14 that maps to the same region of LG14 as PdR1 from b43-17. The geographic separation and different appearance of these plants suggest that this resistance that maps similarly may be different and we are pursuing PdR1c as a future breeding resource. Table 14b summarizes the 12 crosses from this background. The 08-331 set bypasses the potential impact of V. rupestris on resistance, which is present in the 09-367 set. Both are being retained until we more fully understand the inheritance of PD resistance from b40-14. Table 14c is a remake of the 86% viniferaPdR1b x b42-26 line pyramiding cross made last year. These seeds will increase the population size should greenhouse screen results from the initial group prove promising. In the BD5-117 resistance line we see definite differences in Xf titer in backcrosses to vinifera,but the levels are never as low as our most resistant PdR1 individuals. We have created a group of selfed and intercrossed individuals with very good wine variety appearance to advance this resistance source. We also are advancing a very promising source of resistance from V. shuttleworthii, with 08-365 an unusual individual that is as resistant as our most resistant PdR1 genotypes (Table 14d). We have selfed one such particularly resistant BC1 individual to find out whether there is any opportunity to exploit this resistance source (Table 14e). The PD resistance in BD5-117 descends in part from V. shuttleworthii. Three crosses were also made to combine resistance from V. shuttleworthii and BD5-117 (Table 14f).
Table 15 provides a list of PD GH screens initiated and\or completed over the last year. Group A tests genotypes in which PdR1a and PdR1b alleles are combined; results are due soon. Group B tested BC1 and BC2 progeny and their parents in the Haines City V. shuttleworthii line. Results from this screen were used to select the resistant parents for crosses shown in Tables 14e and 14f. Of the 54 accessions tested in the V. arizonica Group C, we selected 5 highly PD resistant individuals from 4 diverse geographic locations for new mapping crosses made in 2012. A table of crosses and estimated seeds has been provided there (Table 16). Results from group D, were used to select the three PD resistant rootstocks advanced for possible release (Table 17). Group E lists further efforts with resistance from M. rotundifolia and is in conjunction with studies of this group’s powdery mildew resistance. Group F tests the last of our elite 94% PdR1b genotypes and the most best looking progeny from parents used in crosses in Tables 14d and 14f and repeated greenhouse screening for the five 94% viniferaPdR1b genotypes advanced for possible release (Table 17). Our 97% vinifera level PdR1b resistance line fruited for the first time in 2011. The phenotypically most promising selections were tested in Group G. This was an extremely severe test and the 9 genotypes were highly resistant and will be advanced to larger scale wine testing. Group H is the third greenhouse screen of a subset of the b42-26 (multi-genic PD resistance) mapping population. Genetic markers in this line are essential as we try to pyramid resistance lines as in the crosses made in Table 14c.
Table 17 lists promising PD resistant selections being considered for release. The release process involves passing a severe greenhouse screen multiple times. The number following the “R” in the “# GH Screens” column notes how many times a selection has passed a severe greenhouse screen. These selections also have desirable viticultural traits and have potential for quality wine production. The production of small lot wines from multiple vines field trials in Davis and in PD hot spots in North Coast complete the evaluation process prior to commercial scale wine lots. PD resistant scions need PD resistant rootstocks: 03300-099; 08314-15; and 08314-46. Screening has found that the latter two have very good nematode resistance as well as PD resistance.
We inoculated our Beringer field trial this year on 5/23/2012. If the favorable growing conditions continue we plan to make two small wine lots from this trial in fall 2012.
Publications
Doucleff, M., Y. Jin and M.A. Walker. 2003. Mapping Xiphinema index resistance in V. rupestris x M. rotundifolia hybrids. ActaHorticulturae 603:79-81.
Krivanek, A.F. and M.A. Walker. 2003. Progress in understanding the genetics of resistance from the grape Muscadinia rotundifolia to the bacterial pathogen Xylella fastidiosa. ActaHorticulturae 603:429-432.
Buzkan, N., L. Kocsis, A.F. Krivanek, and M.A. Walker. 2003. Developing rapid evaluations for resistance to Xylella fastidiosa, the causal agent of Pierce’s disease. ActaHorticulturae 603:433-440.
Buzkan, N. A.F. Krivanek, A. Eskalen and M.A. Walker. 2003. Improvements in sample preparation and polymerase chain reaction detection techniques for Xylella fastidiosa in grapevine tissue. American Journal of Enology and Viticulture 54:307-312.
Doucleff, M, Y. Jin, F. Gao, S. Riaz, A.F. Krivanek and M.A. Walker. 2004. A genetic linkage map of grape utilizing Vitis rupestris and Vitis arizonica. Theoretical and Applied Genetics 109:1178-1187.
Buzkan, N. L. Kocsis and M.A. Walker. 2005. Detection of Xylella fastidiosa from resistant and susceptible grapevine by tissue sectioning and membrane entrapment immunofluorescence. Microbiological Research 160:225-231.
Krivanek, A.F., J.F. Stevenson and M.A. Walker. 2005. Development and comparison of symptom indices for quantifying grapevine resistance to Pierce’s disease. Phytopathology 95:36-43.
Krivanek, A.F. and M.A. Walker. 2005. Vitis resistance to Pierce’s disease is characterized by differential Xylella fastidiosa populations in stems and leaves. Phytopathology 95:44-52.